This project will investigate the mechanism of paraquat cytotoxicity. Despite a consensus that oxygen and the redox properties of paraquat are involved, the ultimate cytotoxic event and the protective role of individual anti-oxidant enzymes have not been identified. This project will investigate these events by a novel approach. The ability of anti- oxidant enzymes to protect against paraquat cytotoxicity will be studied on an individual basis. This will be accomplished by coupling the cDNA of these enzymes to suitable expression vectors. Anti-oxidant enzymes to be studied include copper-zinc superoxide dismutase, manganese superoxide dismutase, glutathione peroxidase, catalase, metallothionein, and glutathione transferases. Two different, selectable, expression vectors will be constructed for each enzyme. One vector will use the noninducible SV promoter, and the other will use the inducible mouse metallothionein (mMT). Preliminary results with a copper-zinc superoxide dismutase expression vector [SV-CuZnSOD-SVneo] indicate the enzyme is produced in large quantities and paraquat resistance develops. The increase in resistance does not correlate to superoxide dismutase activity, but to increase activity of each enzyme against paraquat will be compared to others to determine which enzyme is most effective. The induction of the anti-oxidant enzymes copper-zinc superoxide dismutase, manganese superoxide dismutase, and catalase by paraquat will be studied to determine if the increase occurs by gene amplification, alterations in mRNA, or protein synthesis. In contrast to these anti-oxidant enzymes, glutathione peroxidase appears to be selectively inhibited by paraquat. The mechanism of cell line. This cell line appears to be resistant to paraquat on the basis of increased glutathione peroxidase activity, and not other anti-oxidant enzymes (such as superoxide dismutase), again suggesting that glutathione peroxidase plays an important role in detoxifying paraquat. The mechanism and cellular level (DNA, RNA, protein synthesis) by which this cell continues to express glutathione peroxidase, even when exposed to paraquat, will be determined.